For several years, we have focused on developing much-needed effective, sensitive, and reliable radioligands capable of imaging neuroinflammation. These efforts included the development of now widely-used radioligands for translocator protein (TSPO), as described in past annual reports. Building on this work, we directed our efforts towards the cyclooxygenase (COX) system, which is implicated in the pathophysiology of brain diseases, including Alzheimers Disease and major depressive disorder, and is another potential biomarker for neuroinflammation. The COX system comprises two isoforms, COX-1 and COX-2, which are key enzymes in neuroinflammation. As described in past annual reports, we developed two novel radioligands11CPS13 for COX-1 and 11CMC1 for COX-2that are potent and selective for each isoform and have shown promising results in nonhuman primates. 1) 11CMC1 is the first positron emission tomography (PET) radioligand to successfully image and quantify cyclooxygenase 2 (COX-2) upregulation in living nonhuman primate brain after lipopolysaccharide (LPS)-induced neuroinflammation. Before we undertook this work, PET imaging of neuroinflammation had largely been restricted to studies of TSPO. However, as a biomarker of neuroinflammation, TSPO is limited because it is highly expressed both in activated microglia (the major source of inflammatory mediators) as well as in reactive astrocytes (which can form scars and merely mark sites of former damage and inflammation). For this reason, we sought to develop a PET radioligand for COX-1, which is preferentially located in activated microglia. As reported in a previous annual report, our results demonstrated that 11CPS13 had excellent imaging properties for selectively measuring COX-1 in brain and periphery. Brain uptake was high and reversible; the density of the target enzyme could be stably measured as VT; and about 60-70% of radioligand uptake at baseline reflected COX-1. Compared to 11CKTP-Methe radioligand currently in use11CPS13 had much greater selectivity for COX-1 than COX-2 and did not require synthesis of a pro-drug for entry into brain. Thus, our initial work in this area established that 11CPS13 is a promising radioligand for selectively measuring COX-1 as a biomarker of neuroinflammation and as a potential target for NSAID therapy. Furthermore, uptake of the radioligand in peripheral organs was higher than expected and showed robust blockade (consistent with selective binding to COX-1); this high specific signal in peripheral organs allowed whole body imaging to evaluate the utility of this new radioligand both in brain and in the periphery in a single imaging session. Building on this work, we then sought to determine whether 11CMC1 could image and quantify COX-2 after its upregulation by either one or two intracerebral injections of LPS (a potent inflammogen used here as a model of neuroinflammation). Our study examined COX-2 expression in the brain of four rhesus macaques using the novel PET tracer 11CMC1 at baseline and after injection of LPS into the right putamen. Monkeys 1 and 2 had one LPS injection, and monkeys 3 and 4 had a second injection 33-41 days after the first LPS injection. As a comparator, COX-1 expression was measured using 11CPS13. One and three days after the first LPS injection, COX-1 binding was unchanged. In contrast, COX-2 binding, expressed as the ratio of specific to nondisplaceable uptake (BPND), increased on Day 1 post-LPS injection. A PET scan on the day after the second LPS injection showed a brain lesion (0.5 cm in diameter) that had a high density of COX-2 and a high BPND of 1.8. To verify and extend our PET results, the postmortem brains of these monkeys were histologically examined to identify the density and localization of COX-1 and COX-2 at the gene transcript or protein levels. Postmortem analysis of the brain at the gene transcript and protein level confirmed the in vivo PET results. Moreover, an incidental finding in an unrelated monkey found a line of COX-2 positivity along an incision in skull muscle, demonstrating that 11CMC1 can localize inflammation peripheral to the brain. Taken together, these results show that 11CMC1 is the first PET radioligand to successfully image and quantify COX-2 upregulation in living nonhuman primate brain after LPS-induced neuroinflammation. Notably, 11CPS13 and 11C-MC1 are the first radioligands for COX-1 and COX-2, respectively, that act directly at these targets. In tandem, these ligands could be used to measure COX-1 in healthy conditions and COX-2 in inflammatory disorders, and could also be used to assess drug delivery and in vivo selectivity of NSAIDs in therapeutic trials.